Cellular origins and lineage pathways involved in the development of hepatomas will be investigated in chemically-induced hepatocarcinoma rat models. The specific aims are to determine whether stem cells reside within bile ductules, to characterize surface, nuclear and cytoplasmic properties of stem cells and to determine lineage pathways of stem cells. Specifically are bile ductule epithelial/oval cells the progeny of stem cells and do biliary stem cells differentiate along a bile ductule/oval/transitional cell/preneoplastic nodules/hepatoma pathway. At 2-3 days after the partial hepatectomy (PH) step of the Soft-Farber protocol, we found a putative stem cell compartment within proliferating bile ductules. This compartment contained cells with the morphologic properties of hematopoietic pluripotential stem cells. The liver stem- like cells appear blastlike, have a high nuclear to cytoplasmic ratio, range in size from 3.0-5.0 micro M and are located at the basal aspect of the ductule. The stem cells are completely enclosed by ductule cells and do not exhibit oval cell or bile ductule epithelial phenotypes. The biliary stem-like cell compartment will be analyzed in livers of rats 4-12 hrs. and 1-7 days after PH step of the Solt-Farber protocol for plasma membrane, nuclear, cytoplasmic and proliferation markers (i.e. cytokeratin 19, oval cell antigens, gap junction proteins, actin, sugar- specific lectins, nucleolar organizing regions, 5-bromodeoxyuridine incorporation, desmosome proteins, extra-cellular matrix proteins and ultrastructural properties). These studies will determine: 1) the earliest time after PH when biliary stem cells appear; 2) the presence of intermediary differentiation stages of stem cells; 3) the acquisition by stem cells of polarity, communication structures, cytoskeleton or antigenic properties characteristic of oval/bile ductule epithelial cells and transitional cells; and 4) the relation of biliary stem cells to periductal stem cells. Retroviral genetic labeling of periductal stem cells will be attempted by introducing a marker gene (E. coli- beta galactosidase lac Z gene coupled to a nuclear localization signal) into livers and analyzing with time after PH step of the Soft-Farber protocol, the expression of nuclear beta-galactosidase activity in periductal cells, biliary cells and nodules. These studies will determine the cell lineage pathway of periductal stem cells. Diverse in situ methods will be employed including light, confocal and electron microscopy in conjunction with enzyme-, immuno- and lectin cytochemistry. An in situ approach is expected to reveal new information on a carcinogen-activated liver stem cell compartment and cell lineage pathways not possible by other methodologies. Furthermore, characterization of the stem cell compartment will provide essential information for future studies on the isolation of liver stem cells.